GAL1-3HA-ERB1 cells containing plasmids expressing wild-type or mutant erb1 protein from the pOBD2 vector were grown to an OD600 of 0.6, washed with PBS and treated with 100 mM 2-methyl nicotinic acid imidazolide (NAI) for 20 min at 30°C. Total RNA was extracted with acid phenol:chloroform:isoamyl alcohol (Ambion). Primer extensions of 5 μg of total RNA extracted from NAI-treated cells were performed using Transcriptor Reverse Transcriptase (Roche Diagnostics) with oligonucleotides designed to base-pair with the ITS2 sequence in 27S pre-rRNA (20 (link)).
Protocol detail
NAI-Mediated Pre-rRNA Structural Analysis
GAL1-3HA-ERB1 cells containing plasmids expressing wild-type or mutant erb1 protein from the pOBD2 vector were grown to an OD600 of 0.6, washed with PBS and treated with 100 mM 2-methyl nicotinic acid imidazolide (NAI) for 20 min at 30°C. Total RNA was extracted with acid phenol:chloroform:isoamyl alcohol (Ambion). Primer extensions of 5 μg of total RNA extracted from NAI-treated cells were performed using Transcriptor Reverse Transcriptase (Roche Diagnostics) with oligonucleotides designed to base-pair with the ITS2 sequence in 27S pre-rRNA (20 (link)).
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Acid phenol
Base pair
Cells
Chloroform
Diagnostics
Isoamyl alcohol
Mutant protein
Nicotinic acid
Oligonucleotides
Plasmids
Pre rrna
Primer
Reverse transcriptase
Vector
Corresponding Organization : Carnegie Mellon University
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Variable analysis
independent variables
- Expression of wild-type or mutant erb1 protein from the pOBD2 vector
- Primer extensions of 5 μg of total RNA extracted from NAI-treated cells
- GAL1-3HA-ERB1 cells
- Growth of cells to an OD600 of 0.6
- Washing of cells with PBS
- Treatment of cells with 100 mM 2-methyl nicotinic acid imidazolide (NAI) for 20 min at 30°C
- Total RNA extraction with acid phenol:chloroform:isoamyl alcohol (Ambion)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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